Plant Chloroplast Genomes Research Articles

Characterization of the DNA accessibility of chloroplast genomes in grasses

Although the chloroplast genome (cpDNA) of higher plants is known to exist as a large protein-DNA complex called ‘plastid nucleoid’, researches on its DNA state and regulatory elements are limited. In this study, we performed the assay for transposase-accessible chromatin sequencing (ATAC-seq) on five common tissues across five grasses, and found that the accessibility of different regions in cpDNA varied widely, with the transcribed regions being highly accessible and accessibility patterns around gene start and end sites varying depending on the level of gene expression. Further analysis identified a total of 3970 putative protein binding footprints on cpDNAs of five grasses. These footprints were enriched in intergenic regions and co-localized with known functional elements. Footprints and their flanking accessibility varied dynamically among tissues. Cross-species analysis showed that footprints in coding regions tended to overlap non-degenerate sites and contain a high proportion of highly conserved sites, indicating that they are subject to evolutionary constraints. Taken together, our results suggest that the accessibility of cpDNA has biological implications and provide new insights into the transcriptional regulation of chloroplasts.

Removal of the large inverted repeat from the plastid genome reveals gene dosage effects and leads to increased genome copy number

The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.

The complete chloroplast genome sequence of Vitis vinifera ‘Krasnostop Zolotovskiy’, an autochthonous variety of the Don Valley

DNA sequence data from complete chloroplast genomes expand our understanding of plant diversity and contributes to our understanding of evolutionary relationships among species. De novo assembly and annotation of plant chloroplast genomes are difficult due to the high number of repeats and low number of plant chloroplast genomes in international databases. The development of long-read sequencing platforms such as PacBio and Oxford Nanopore, as well as specialized assemblers, is simplifying the process of assembling and annotating large circular molecules. In this work, we report the complete chloroplast genome of the autochthonous Vitis vinifera variety ‘Krasnostop Zolotovskiy’, obtained de novo by the hybrid assembly method using short and long DNA reads. The circular genome of the chloroplast is 160,927 bp long with a GC ratio of 37.38 %. It has four subregions: large single-copy (LSC) region of 71,946 bp, a small single-copy (SSC) region of 36,273 bp, and two inverted repeat regions (IRs) of 26,354 bp each. The chloroplast genome of V. vinifera ‘Krasnostop Zolotovskiy’ harbours 129 genes, comprising 84 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. We also carry out a phylogenetic analysis based on all the complete sequences of grape chloroplast genomes available in the databases, which shows that the V. vinifera ‘Krasnostop Zolotovskiy’ does not have a direct origin with any European variety.

Phylogenetic relationships, selective pressure and molecular markers development of six species in subfamily Polygonoideae based on complete chloroplast genomes

The subfamily Polygonoideae encompasses a diverse array of medicinal and horticultural plants that hold significant economic value. However, due to the lack of a robust taxonomy based on phylogenetic relationships, the classification within this family is perplexing, and there is also a scarcity of reports on the chloroplast genomes of many plants falling under this classification. In this study, we conducted a comprehensive analysis by sequencing and characterizing the complete chloroplast genomes of six Polygonoideae plants, namely Pteroxygonum denticulatum, Pleuropterus multiflorus, Pleuropterus ciliinervis, Fallopia aubertii, Fallopia dentatoalata, and Fallopia convolvulus. Our findings revealed that these six plants possess chloroplast genomes with a typical quadripartite structure, averaging 162,931 bp in length. Comparative chloroplast analysis, codon usage analysis, and repetitive sequence analysis demonstrated a high level of conservation within the chloroplast genomes of these plants. Furthermore, phylogenetic analysis unveiled a distinct clade occupied by P. denticulatum, while P. ciliinrvis displayed a closer relationship to the three plants belonging to the Fallopia genus. Selective pressure analysis based on maximum likelihood trees showed that a total of 14 protein-coding genes exhibited positive selection, with psbB and ycf1 having the highest number of positive amino acid sites. Additionally, we identified four molecular markers, namely petN-psbM, psal-ycf4, ycf3-trnS-GGA, and trnL-UAG-ccsA, which exhibit high variability and can be utilized for the identification of these six plants.

Cross amplification of 16S rRNA bacterial primer 27F/1492R on horticultural crop chloroplast genome

Molecular techniques have been applied as fast and accurate detection methods of plant pathogenic bacteria. Bacteria-specific primer 27F paired with the universal primer 1492R was used in PCR to detect plant pathogenic bacteria in symptomatic leaves of tomato (Solanum lycopersicum L.), chili pepper (Capsicum annum L.), napa cabbage (Brassica rapa subsp. pekinensis L.), cabbage (Brassica oleracea var. capitata L.), and longan (Dimocarpus longan Lour.). The targeted single bands with length of 1400 bp were obtained but bidirectional sequencing of the PCR products recovered partial sequence of chloroplast instead of bacterial genomes. Thus, this result confirmed cross amplification of 27F/1492R primer pair against chloroplast genome of plants. Phylogenetic tree was constructed to separate the tested isolates according to their taxonomic relations: Order Sapindales, Solanales, and Brassicales. In-silico analysis on the new five and nineteen selected sequences in NCBI GenBank detected at least seven conserved regions with some single nucleotide polymorphisms. This report might be a cautionary advice for other researchers to avoid false positive results which could lead to misdiagnosis of bacterial plant diseases.

The newly assembled chloroplast genome of Aeluropus littoralis: molecular feature characterization and phylogenetic analysis with related species

Aeluropus littoralis, a halophyte grass, is widely distributed from the Mediterranean to the Indian subcontinent through the Mongolian Gobi. This model halophyte has garnered increasing attention owing to its use as forage and its high tolerance to environmental stressors. The chloroplast genomes of many plants have been extensively examined for molecular, phylogenetic and transplastomic applications. However, no published research on the A. littoralis chloroplast (cp) genome was discovered. Here, the entire chloroplast genome of A. littoralis was assembled implementing accurate long-read sequences. The entire chloroplast genome, with an estimated length of 135,532 bp (GC content: 38.2%), has a quadripartite architecture and includes a pair of inverted repeat (IR) regions, IRa and IRb (21,012 bp each), separated by a large and a small single-copy regions (80,823 and 12,685 bp, respectively). The features of A. littoralis consist of 133 genes that synthesize 87 peptides, 38 transfer RNAs, and 8 ribosomal RNAs. Of these genes, 86 were unique, whereas 19 were duplicated in IR regions. Additionally, a total of forty-six simple sequence repeats, categorized into 32-mono, four-di, two-tri, and eight-tetranucleotides, were discovered. Furthermore, ten sets of repeats greater than 20 bp were located primarily in the LSC region. Evolutionary analysis based on chloroplast sequence data revealed that A. littoralis with A. lagopoides and A. sinensis belong to the Aeluropodinae subtribe, which is a sister to the Eleusininae in the tribe Cynodonteae and the subfamily Chloridoideae. This subfamily belongs to the PACMAD clade, which contains the majority of the C4 photosynthetic plants in the Poaceae. The newly constructed A. littoralis cp genome offers valuable knowledge for DNA barcoding, phylogenetic, transplastomic research, and other biological studies.

Both-strand gene coding in a plastome-like mitogenome of an enoplid nematode.

The phylum Nematoda remains very poorly sampled for mtDNA, with a strong bias toward parasitic, economically important or model species of the Chromadoria lineage. Most chromadorian mitogenomes share a specific order of genes encoded on one mtDNA strand. However, the few sequenced representatives of the Dorylaimia lineage exhibit a variable order of mtDNA genes encoded on both strands. While the ancestral arrangement of nematode mitogenome remains undefined, no evidence has been reported for Enoplia, the phylum's third early divergent major lineage. We describe the first mitogenome of an enoplian nematode, Campydora demonstrans, and contend that the complete 37-gene repertoire and both-strand gene encoding are ancestral states preserved in Enoplia and Dorylaimia versus the derived mitogenome arrangement in some Chromadoria. The C. demonstrans mitogenome is 17,018 bp in size and contains a noncoding perfect inverted repeat with 2013 bp-long arms, subdividing the mitogenome into two coding regions. This mtDNA arrangement is very rare among animals and instead resembles that of chloroplast genomes in land plants. Our report broadens mtDNA taxonomic sampling of the phylum Nematoda and adds support to the applicability of cox1 gene as a phylogenetic marker for establishing nematode relationships within higher taxa.

Comparison of the Complete Chloroplast Genomes in the Astragalus

Background: Astragalus is one of the largest angiosperms and has important economic value. The chloroplast genomes (Cp) of most plants in its family have been sequenced and annotated, but there are fewer studies on the characteristics of the chloroplast genomes and codon usage bias of Astragalus. In this study, we sequenced and annotated the complete chloroplast genomes of three Astragalus species, that systematically compared the chloroplast genome and codon usage characteristics of three Astragalus. Methods: In this study, we used three Astragalus species as materials. Firstly, we sequenced and assembled their chloroplast genomes. Subsequently, we analyzed the codon usage bias of the chloroplast genomes of the three medicinal Astragalus species using software such as CodonW, CUSP and SPSS. Result: The results illustrated that the length of Astragalus chloroplast genome ranges from 122,815 bp (A. dahuricus) to 123,729 bp (A. melilotoides Pall.). There were 107-110genes, including 75-76 protein-coding genes (PCGs), four ribosomal RNA genes (rRNAs) and 28-30 transfer RNA genes (tRNAs). In addition, the results illustrated that codons in three Cp genomes from Astragalus had the same 11 Optimal codons and ending with A/U. The codon usage frequency of five model creatures were compared and the results revealed that the codon preferences of Astragalus and five model creatures were pretty significant, the natural selection was the main factor in codon preference. Our research provides some value for the researches of chloroplast genetic engineering and molecular breeding in Astragalus.

Genome Editing of Plant Mitochondrial and Chloroplast Genomes.

Plastids (including chloroplasts) and mitochondria are remnants of endosymbiotic bacteria, yet they maintain their own genomes, which encode vital components for photosynthesis and respiration, respectively. Organellar genomes have distinctive features, such as being present as multicopies, being mostly inherited maternally, having characteristic genomic structures and undergoing frequent homologous recombination. To date, it has proven to be challenging to modify these genomes. For example, while CRISPR/Cas9 is a widely used system for editing nuclear genes, it has not yet been successfully applied to organellar genomes. Recently, however, precise gene-editing technologies have been successfully applied to organellar genomes. Protein-based enzymes, especially transcription activator-like effector nucleases (TALENs) and artificial enzymes utilizing DNA-binding domains of TALENs (TALEs), have been successfully used to modify these genomes by harnessing organellar-targeting signals. This short review introduces and discusses the use of targeted nucleases and base editors in organellar genomes, their effects and their potential applications in plant science and breeding.

Current state of research in the development of the genomic editing method: problems and prospects

The possibility of using the CRISPR/Cas method of genomic editing has provided researchers with a powerful tool not only for targeted modification of genes that determine economically valuable traits in plants, but also for solving fundamental problems of their functioning. The most striking examples of the use of CRISPR/Cas9 to improve various plant species by knockouts of target genes or knockins of expression cassettes, including genes that change the biosynthesis of important plant metabolites, obtained by foreign research groups, are presented. We discuss our own results on the directed change in the functioning of genes encoding photosystem II carbonic anhydrases, as well as genes involved in plant responses to stress in theArabidopsis thalianamodel. Examples of the use of the genomic editing method to improve the characteristics of plant cell cultures as bioproducers of pharmaceutically valuable recombinant proteins are given. Methodological issues related to plant genome editing are considered — the problems of chimerism, obtaining homozygotes and biallelic knockout mutations, knockout of regulatory and structural genes, as well as repair features in the regions of integration of expression cassettes in knockins. The main directions for further development and improvement of the CRISPR/Cas genomic editing method aimed at optimizing the efficiency of delivery of target genetic constructs and editing tools to the nuclear and chloroplast genomes of plants using single-walled carbon nanotubes are summarized.

Relocation of chloroplast proteins from cytosols into chloroplasts

ABSTRACT The chloroplasts in terrestrial plants play a functional role as a major sensor for perceiving physiological changes under normal and stressful conditions. Despite the fact that the plant chloroplast genome encodes around 120 genes, which are mainly essential for photosynthesis and chloroplast biogenesis, the functional roles of the genes remain to be determined in plant’s response to environmental stresses. Photosynthetic electron transfer D (PETD) is a key component of the chloroplast cytochrome b6f complex. Chloroplast ndhA (NADH dehydrogenase A) and ndhB (NADH dehydrogenase B) interact with photosystem I (PSI), forming NDH-PSI supercomplex. Notably, artificial targeting of chloroplasts-encoded proteins, PETD, NDHA, or NDHB, was successfully relocated from cytosols into chloroplasts. The result suggests that artificial targeting of proteins to chloroplasts is potentially open to the possibility of chloroplast biotechnology in engineering of plant tolerance against biotic and abiotic stresses.

Complete chloroplast genome of Lens lamottei reveals intraspecies variation among with Lens culinaris

Lens lamottei is a member of the Fabaceae family and the second gene pool of the genus Lens. The environmental factors that drove the divergence among wild and cultivated species have been studied extensively. Recent research has focused on genomic signatures associated with various phenotypes with the acceleration of next-generation techniques in molecular profiling. Therefore, in this study, we provide the complete sequence of the chloroplast genome sequence in the wild Lens species L. lamottei with a deep coverage of 713 × next-generation sequencing (NGS) data for the first time. Compared to the cultivated species, Lens culinaris, we identified synonymous, and nonsynonymous changes in the protein-coding regions of the genes ndhB, ndhF, ndhH, petA, rpoA, rpoC2, rps3, and ycf2 in L. lamottei. Phylogenetic analysis of chloroplast genomes of various plants under Leguminosae revealed that L. lamottei and L. culinaris are closest to one another than to other species. The complete chloroplast genome of L. lamottei also allowed us to reanalyze previously published transcriptomic data, which showed high levels of gene expression for ATP-synthase, rubisco, and photosystem genes. Overall, this study provides a deeper insight into the diversity of Lens species and the agricultural importance of these plants through their chloroplast genomes.

An analysis of codon utilization patterns in the chloroplast genomes of three species of Coffea

BackgroundThe chloroplast genome of plants is known for its small size and low mutation and recombination rates, making it a valuable tool in plant phylogeny, molecular evolution, and population genetics studies. Codon usage bias, an important evolutionary feature, provides insights into species evolution, gene function, and the expression of exogenous genes. Coffee, a key crop in the global tropical agricultural economy, trade, and daily life, warrants investigation into its codon usage bias to guide future research, including the selection of efficient heterologous expression systems for coffee genetic transformation.ResultsAnalysis of the codon utilization patterns in the chloroplast genomes of three Coffea species revealed a high degree of similarity among them. All three species exhibited similar base compositions, with high A/T content and low G/C content and a preference for A/T-ending codons. Among the 30 high-frequency codons identified, 96.67% had A/T endings. Fourteen codons were identified as ideal. Multiple mechanisms, including natural selection, were found to influence the codon usage patterns in the three coffee species, as indicated by ENc-GC3s mapping, PR2 analysis, and neutral analysis. Nicotiana tabacum and Saccharomyces cerevisiae have potential value as the heterologous expression host for three species of coffee genes.ConclusionThis study highlights the remarkable similarity in codon usage patterns among the three coffee genomes, primarily driven by natural selection. Understanding the gene expression characteristics of coffee and elucidating the laws governing its genetic evolution are facilitated by investigating the codon preferences in these species. The findings can enhance the efficacy of exogenous gene expression and serve as a basis for future studies on coffee evolution.

Genomic Analysis of Plastid-Nuclear Interactions and Differential Evolution Rates in Coevolved Genes across Juglandaceae Species.

The interaction between the nuclear and chloroplast genomes in plants is crucial for preserving essential cellular functions in the face of varying rates of mutation, levels of selection, and modes of transmission. Despite this, identifying nuclear genes that coevolve with chloroplast genomes at a genome-wide level has remained a challenge. In this study, we conducted an evolutionary rate covariation analysis to identify candidate nuclear genes coevolving with chloroplast genomes in Juglandaceae. Our analysis was based on 4,894 orthologous nuclear genes and 76 genes across seven chloroplast partitions in nine Juglandaceae species. Our results indicated that 1,369 (27.97%) of the nuclear genes demonstrated signatures of coevolution, with the Ycf1/2 partition yielding the largest number of hits (765) and the ClpP1 partition yielding the fewest (13). These hits were found to be significantly enriched in biological processes related to leaf development, photoperiodism, and response to abiotic stress. Among the seven partitions, AccD, ClpP1, MatK, and RNA polymerase partitions and their respective hits exhibited a narrow range, characterized by dN/dS values below 1. In contrast, the Ribosomal, Photosynthesis, Ycf1/2 partitions and their corresponding hits, displayed a broader range of dN/dS values, with certain values exceeding 1. Our findings highlight the differences in the number of candidate nuclear genes coevolving with the seven chloroplast partitions in Juglandaceae species and the correlation between the evolution rates of these genes and their corresponding chloroplast partitions.

Comparative analysis of 17 complete chloroplast genomes reveals intraspecific variation and relationships among Pseudostellaria heterophylla (Miq.) Pax populations.

Pseudostellaria heterophylla (Miq.) Pax is a well-known medicinal and ecologically important plant. Effectively distinguishing its different genetic resources is essential for its breeding. Plant chloroplast genomes can provide much more information than traditional molecular markers and provide higher-resolution genetic analyses to distinguish closely related planting materials. Here, seventeen P. heterophylla samples from Anhui, Fujian, Guizhou, Hebei, Hunan, Jiangsu, and Shandong provinces were collected, and a genome skimming strategy was employed to obtain their chloroplast genomes. The P. heterophylla chloroplast genomes ranged from 149,356 bp to 149,592 bp in length, and a total of 111 unique genes were annotated, including 77 protein-coding genes, 30 tRNA genes, and four rRNA genes. Codon usage analysis showed that leucine had the highest frequency, while UUU (encoding phenylalanine) and UGC (encoding cysteine) were identified as the most and least frequently used codons, respectively. A total of 75-84 SSRs, 16-21 short tandem repeats, and 27-32 long repeat structures were identified in these chloroplast genomes. Then, four primer pairs were revealed for identifying SSR polymorphisms. Palindromes are the dominant type, accounting for an average of 47.86% of all long repeat sequences. Gene orders were highly collinear, and IR regions were highly conserved. Genome alignment indicated that there were four intergenic regions (psaI-ycf4, ycf3-trnS, ndhC-trnV, and ndhI-ndhG) and three coding genes (ndhJ, ycf1, and rpl20) that were highly variable among different P. heterophylla samples. Moreover, 10 SNP/MNP sites with high polymorphism were selected for further study. Phylogenetic analysis showed that populations of Chinese were clustered into a monophyletic group, in which the non-flowering variety formed a separate subclade with high statistical support. In this study, the comparative analysis of complete chloroplast genomes revealed intraspecific variations in P. heterophylla and further supported the idea that chloroplast genomes could elucidate relatedness among closely related cultivation materials.

Comparative chloroplast genome analysis of Sambucus L. (Viburnaceae): inference for phylogenetic relationships among the closely related Sambucus adnata Wall. ex DC Sambucus javanica Blume.

Sambucus L. is found in the family Viburnaceae (syn. Adoxaceae) and encompasses approximately 29 accepted species. The complex morphology of these species has caused continued confusion concerning their nomenclature, classification, and identification. Despite previous attempts to resolve taxonomic complexities in the Sambucus genus, there are still unclear phylogenetic relationships among several species. In this study, the newly obtained plastome of Sambucus williamsii Hance. as well as the populations of Sambucus canadensis L., Sambucus javanica Blume, and Sambucus adnata Wall. ex DC were sequenced, and their sizes, structural similarity, gene order, gene number, and guanine-cytosine (GC) contents were analyzed. The phylogenetic analyses were conducted using the whole chloroplast genomes and protein-coding genes (PCGs). The findings revealed that the chloroplast genomes of Sambucus species exhibited typical quadripartite double-stranded DNA molecules. Their lengths ranged from 158,012 base pairs (bp) (S. javanica) to 158,716 bp (S. canadensis L). Each genome comprised a pair of inverted repeats (IRs), which separated the large single-copy (LSC) and small single-copy (SSC) regions. In addition, the plastomes contained 132 genes, encompassing 87 protein-coding, 37 tRNA, and four rRNA genes. In the simple sequence repeat (SSR) analysis, A/T mononucleotides had the highest proportion, with the most repetitive sequences observed in S. williamsii. The comparative genome analyses showed high similarities in structure, order, and gene contents. The hypervariable regions in the studied chloroplast genomes were trnT-GGU, trnF-GAA, psaJ, trnL-UAG, ndhF, and ndhE, which may be used as candidate barcodes for species discrimination in Sambucus genus. Phylogenetic analyses supported the monophyly of Sambucus and revealed the separation of S. javanica and S. adnata populations. Sambucus chinensis Lindl. was nested within S. javanica in the same clade, collaborating their conspecific treatment. These outcomes indicate that the chloroplast genome of Sambucus plants is a valuable genetic resource for resolving taxonomic discrepancies at the lower taxonomic levels and can be applied in molecular evolutionary studies.

CPJSdraw: analysis and visualization of junction sites of chloroplast genomes.

Chloroplast genomes are usually circular molecules, and most of them are tetrad structures with two inverted repeat (IR) regions, a large single-copy region, and a small single-copy region. IR contraction and expansion are among the genetic diversities during the evolution of plant chloroplast genomes. The only previously released tool for the visualization of junction sites of the regions does not consider the diversity of the starting point of genomes, which leads to incorrect results or even no results for the examination of IR contraction and expansion. In this work, a new tool named CPJSdraw was developed for visualizing the junction sites of chloroplast genomes. CPJSdraw can format the starting point of the irregular linearized genome, correct the junction sites of IR and single-copy regions, display the tetrad structure, visualize the junction sites of any number (≥1) of chloroplast genomes, show the transcription direction of genes adjacent to junction sites, and indicate the IR expansion or contraction of chloroplast genomes. CPJSdraw is a software that is universal and reliable in analysis and visualization of IR expansion or contraction of chloroplast genomes. CPJSdraw has more accurate analysis and more complete functions when compared with previously released tool. CPJSdraw as a perl package and tested data are available at http://dx.doi.org/10.5281/zenodo.7669480 for English users. In addition, an online version with a Chinese interface is available at http://cloud.genepioneer.com:9929/#/tool/alltool/detail/335.

Gene Losses and Homology of the Chloroplast Genomes of Taxillus and Phacellaria Species.

Research on the chloroplast genome of parasitic plants is limited. In particular, the homology between the chloroplast genomes of parasitic and hyperparasitic plants has not been reported yet. In this study, three chloroplast genomes of Taxillus (Taxillus chinensis, Taxillus delavayi, and Taxillus thibetensis) and one chloroplast genome of Phacellaria (Phacellaria rigidula) were sequenced and analyzed, among which T. chinensis is the host of P. rigidula. The chloroplast genomes of the four species were 119,941-138,492 bp in length. Compared with the chloroplast genome of the autotrophic plant Nicotiana tabacum, all of the ndh genes, three ribosomal protein genes, three tRNA genes and the infA gene were lost in the three Taxillus species. Meanwhile, in P. rigidula, the trnV-UAC gene and the ycf15 gene were lost, and only one ndh gene (ndhB) existed. The results of homology analysis showed that the homology between P. rigidula and its host T. chinensis was low, indicating that P. rigidula grows on its host T. chinensis but they do not share the chloroplast genome. In addition, horizontal gene transfer was not found between P. rigidula and its host T. chinensis. Several candidate highly variable regions in the chloroplast genomes of Taxillus and Phacellaria species were selected for species identification study. Phylogenetic analysis revealed that the species of Taxillus and Scurrula were closely related and supported that Scurrula and Taxillus should be treated as congeneric, while species in Phacellaria had a close relationship with that in Viscum.

Anticodon table of the chloroplast genome and identification of putative quadruplet anticodons in chloroplast tRNAs

The chloroplast genome of 5959 species was analyzed to construct the anticodon table of the chloroplast genome. Analysis of the chloroplast transfer ribonucleic acid (tRNA) revealed the presence of a putative quadruplet anticodon containing tRNAs in the chloroplast genome. The tRNAs with putative quadruplet anticodons were UAUG, UGGG, AUAA, GCUA, and GUUA, where the GUUA anticodon putatively encoded tRNAAsn. The study also revealed the complete absence of tRNA genes containing ACU, CUG, GCG, CUC, CCC, and CGG anticodons in the chloroplast genome from the species studied so far. The chloroplast genome was also found to encode tRNAs encoding N-formylmethionine (fMet), Ile2, selenocysteine, and pyrrolysine. The chloroplast genomes of mycoparasitic and heterotrophic plants have had heavy losses of tRNA genes. Furthermore, the chloroplast genome was also found to encode putative spacer tRNA, tRNA fragments (tRFs), tRNA-derived, stress-induced RNA (tiRNAs), and the group I introns. An evolutionary analysis revealed that chloroplast tRNAs had evolved via multiple common ancestors and the GC% had more influence toward encoding the tRNA number in the chloroplast genome than the genome size.

Single-molecule Sequencing of an Animal Mitochondrial Genome Reveals Chloroplast-like Architecture and Repeat-mediated Recombination.

Recent advances in long-read sequencing technology have allowed for single-molecule sequencing of entire mitochondrial genomes, opening the door for direct investigation of the mitochondrial genome architecture and recombination. We used PacBio sequencing to reassemble mitochondrial genomes from two species of New Zealand freshwater snails, Potamopyrgus antipodarum and Potamopyrgus estuarinus. These assemblies revealed a ∼1.7 kb structure within the mitochondrial genomes of both species that was previously undetected by an assembly of short reads and likely corresponding to a large noncoding region commonly present in the mitochondrial genomes. The overall architecture of these Potamopyrgus mitochondrial genomes is reminiscent of the chloroplast genomes of land plants, harboring a large single-copy (LSC) region and a small single-copy (SSC) region separated by a pair of inverted repeats (IRa and IRb). Individual sequencing reads that spanned across the Potamopyrgus IRa-SSC-IRb structure revealed the occurrence of a "flip-flop" recombination. We also detected evidence for two distinct IR haplotypes and recombination between them in wild-caught P. estuarinus, as well as extensive intermolecular recombination between single-nucleotide polymorphisms in the LSC region. The chloroplast-like architecture and repeat-mediated mitochondrial recombination we describe here raise fundamental questions regarding the origins and commonness of inverted repeats in cytoplasmic genomes and their role in mitochondrial genome evolution.